Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The bifunctional rat liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) is constructed of two independent catalytic domains. We present evidence that the kinase and bisphosphatase halves of the bifunctional enzyme are, respectively, structurally similar to the glycolytic enzymes 6-phosphofructo-1-kinase and phosphoglycerate mutase. Computer-assisted modeling of the C-terminal bisphosphatase domain reveals a hydrophobic core and active site residue constellation equivalent to the yeast mutase structure; structural differences map to length-variable, surface-located loops. Sequence patterns derived from the structural alignment of mutases and the bisphosphatase further detect a significant similarity to a family of acid phosphatases. The N-terminal kinase domain, in turn, is predicted to form a nucleotide-binding fold that is analogous to a segment of 6-phosphofructo-1-kinase, suggesting that these unrelated enzymes bind fructose 6-phosphate and ATP substrates in a similar geometry. This analysis indicates that the bifunctional enzyme is the likely product of gene fusion of kinase and mutase/phosphatase catalytic units.     

5'' flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis, are catalyzed by 6-phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46), respectively. In liver, these two activities belong to separate domains of the same 470-residue polypeptide. Various mRNAs have been described for this bifunctional enzyme, which is controlled by hormonal and metabolic signals. To understand the origin and regulation of these mRNAs, we have characterized rat genomic clones encoding the liver isozyme, which is regulated by cAMP-dependent protein kinase, and the muscle isozyme, which is not. We describe here a 55-kilobase gene that encodes these isozymes by alternative splicing from two promoters. Each of the putative promoters was sequenced over about 3 kilobases and found to include nucleotide motifs for binding regulatory factors. The two isozymes share the same 13 exons and differ only by the first exon that, in the liver but not in the muscle isozyme, contains the serine phosphorylated by cAMP-dependent protein kinase. The gene was assigned to the X chromosome. An analysis of the exon limits of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in relation to its functional domains and to its similarity with other proteins plus its G + C content at the third codon position suggests that this gene originates from several fusion events.     

Tissue distribution, immunoreactivity, and physical properties of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

6-Phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46) activities were determined in various rat tissues, the latter by using a method based on the formation of a phosphorylated enzyme intermediate during the course of catalysis. Both activities from liver, skeletal muscle, lung, kidney, and testis copurified during polyethylene glycol fractionation, anion-exchange and blue Sepharose chromatography, and gel filtration. The Stokes radius of these enzymes and of the liver bifunctional enzyme was 45 A. Extrahepatic tissues had only 10% or less of the kinase activity found in liver. The results indicate that a liver-type bifunctional enzyme is present in most extrahepatic tissues but that it is minimally expressed. However, the ratio of kinase to bisphosphatase activity in most extrahepatic tissues was 4- to 6-fold higher than in liver, whereas heart 6-phosphofructo-2-kinase had no associated bisphosphatase activity, although its Stokes radius was also 45 A. The heart enzyme was not precipitated by an antiserum to the liver enzyme, whereas only a fraction of the kidney and testis activities was precipitated by this antiserum. The data support the existence of a distinct form of extrahepatic 6-phosphofructo-2-kinase, most readily demonstrated in heart, which may not be bifunctional.     

Complete amino acid sequence of pig kidney fructose-1,6-bisphosphatase.

The covalent structure of the pig kidney fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) subunit has been determined. Placement of the 335 amino acid residues in the polypeptide chain was based largely on automated Edman degradation of eight purified cyanogen bromide fragments generated from the S-carboxymethylated protein. The determination of the amino acid sequence of the largest cyanogen bromide fragment (154 residues) required additional analysis of subfragments obtained by tryptic cleavage at arginyl residues and by mild acid cleavage of an Asp-Pro peptide bond. Alignment of the cyanogen bromide fragments was accomplished by analysis of a product of limited proteolysis by an endogenous protease and by characterization of the tryptic peptides isolated from S-[14C]carboxymethylated fructose-1,6-bisphosphatase. This sequence information has permitted the identification of several reactive sites of functional and structural significance in pig kidney fructose-1,6-bisphosphatase.     

Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli.

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.     

Expression of the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli

The fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.105/EC 3.1.3.46) was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The protein was efficiently expressed (i) as a fusion protein that starts at the T7 major capsid protein initiation site in a pET expression vector and (ii) as a protein that starts within the bisphosphatase sequence by translation reinitiation. Both proteins have similar properties. The protein was purified to homogeneity by anion-exchange chromatography and gel filtration. The purified fructose-2,6-bisphosphatase domain was active and no 6-phosphofructo-2-kinase activity was found associated with it. In contrast to the dimeric bifunctional enzyme, the fructose-2,6-bisphosphatase domain behaved as a monomer of 30 kDa. The turnover number and kinetic properties of the separate bisphosphatase domain were similar to those of the bisphosphatase of the bifunctional enzyme, including the ability to form a phosphoenzyme intermediate. These results support the hypothesis that the rat liver enzyme consists of two independent domains and is a member of a class of enzymes formed by gene fusion.     

Regulation of fructose-6-phosphate 2-kinase by phosphorylation and dephosphorylation: possible mechanism for coordinated control of glycolysis and glycogenolysis.

The kinetic properties and the control mechanism of fructose-6-phosphate 2-kinase (ATP: D-fructose-6-phosphate 2-phosphotransferase) were investigated. The molecular weight of the enzyme is approximately 100,000 as determined by gel filtration. The plot of initial velocity versus ATP concentration is hyperbolic with a Km of 1.2 mM. However, the plot of enzyme activity as a function of fructose-6-phosphate is sigmoidal. The apparent K0.5 for fructose-6-phosphate is 20 microM. Fructose-6-phosphate 2-kinase is inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase, and the inactivation is closely correlated with phosphorylation. The enzyme is also inactivated by phosphorylase kinase in the presence of Ca2+ and calmodulin. The phosphorylated fructose-6-phosphate 2-kinase, which is inactive, is activated by phosphorylase phosphatase and alkaline phosphatase. The possible physiological significance of these observations in the coordinated control of glycogen metabolism and glycolysis is discussed.     

Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase.

I have isolated glucose-6-phosphate dehydrogenase from rabbit liver microsomes and determined its complete amino acid sequence. Sequence determination was achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The microsomal enzyme consists of 763 residues and is quite dissimilar from the previously characterized cytosolic enzymes. The N terminus of the microsomal enzyme is blocked by a pyroglutamyl residue. Carbohydrate is attached at Asn-138 and Asn-263, implying that the bulk of the protein is oriented on the lumenal side of the endoplasmic membrane. The amino acid sequence of the microsomal protein shows limited homology to the extensively sequenced cytosolic glucose-6-phosphate dehydrogenases. Clusters of up to six identical residues can be identified in four regions: peptide segments at residues 10-21, 154-163, and 173-261. In addition, another array of identical residues, requiring a 100-residue deletion in the sequence of the microsomal enzyme, spans residues 436-462 and corresponds to residues 348-373 of the cytosolic protein. Two segments with a Gly-Xaa-Gly-Xaa-Xaa-Gly motif, related to a coenzyme binding fold, were identified at Gly-399 and Gly-491. In the cytosolic enzymes, a variation of this sequence motif occurs at Gly-37 and Gly-241. The 300-residue C-terminal segment of the microsomal enzyme is unique and has no counterpart in the cytosolic or the bacterial enzymes. An unexpected finding with regard to the microsomal enzyme is that it lacks an identifiable membrane-spanning region or the lumenal-protein C-terminal consensus sequences Lys-Asp-Glu or His-Ile/Thr-Glu-Leu. Thus, the mode of transport and retention of this protein in the lumen of endoplasmic reticulum remains to be determined.     

Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 6-phosphate, AMP, and magnesium.

The crystal structure of fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with fructose 6-phosphate, AMP, and Mg2+ has been solved by the molecular replacement method and refined at 2.5-A resolution to a R factor of 0.215, with root-mean-square deviations of 0.013 A and 3.5 degrees for bond lengths and bond angles, respectively. No solvent molecules have been included in the refinement. This structure shows large quaternary and tertiary conformational changes from the structures of the unligated enzyme or its fructose 2,6-bisphosphate complex, but the secondary structures remain essentially the same. Dimer C3-C4 of the enzyme-fructose 6-phosphate-AMP-Mg2+ complex twists about 19 degrees relative to the same dimer of the enzyme-fructose 2,6-bisphosphate complex if their C1-C2 dimers are superimposed on one another. Nevertheless, many interfacial interactions between dimers of C1-C2 and C3-C4 are conserved after quaternary structure changes occur. Residues of the AMP domain (residues 6-200) show large migrations of C alpha atoms relative to barely significant positional changes of the FBP domain (residues 201-335).     

The complete amino acid sequence of prochymosin.

The total sequence of 365 amino acid residues in bovine prochymosin is presented. Alignment with the amino acid sequence of porcine pepsinogen shows that 204 amino acid residues are common to the two zymogens. Further comparison and alignment with the amino acid sequence of penicillopepsin shows that 66 residues are located at identical positions in all three proteases. The three enzymes belong to a large group of proteases with two aspartate residues in the active center. This group forms a family derived from one common ancestor.     

In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.

Incorporation of 32P from [gamma-32P]ATP into a homogenous preparation of rat hepatic fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) was catalyzed by a homogeneous preparation of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine liver. Approximately 4 mol of phosphate were incorporated per mol of the tetrameric enzyme. This phosphorylation was associated with an increase in enzyme activity. In addition, in vivo phosphorylation of the enzyme was observed after injection of radioactive inorganic phosphate into rats and subsequent isolation of the enzyme by conventional purification methods and by immunoprecipitation. All of the labeled phosphate incorporation into the enzyme, both in vitro and in vivo, was precipitated by antibody specific for the enzyme. Furthermore, the 32Pi counts were coincident with the enzyme subunit band when the immunoprecipitates were examined by sodium dodecyl sulfate/disc gel electrophoresis. Acid hydrolysis of the immunoprecipitated enzyme that was phosphorylated in vitro revealed that only seryl residues were labeled. On the basis of the concentration of protein kinase (0.2-1.0 muM) necessary to phosphorylate physiological amounts of fructose-1,6-bisphosphatase (1.0-4.0 muM), it is suggested that cyclic AMP-dependent protein kinase may catalyze the phosphorylation of fructose-1,6-bisphosphatase in vivo.     

Bovine protein C: amino acid sequence of the light chain.

The amino acid sequence of the light chain of bovine protein C was determined by sequenator analysis of the carboxymethylated light chain and fragments obtained by cyanogen bromide treatment, tryptic digestion after blocking of lysine residues, and cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole). The sequence was (in the standard one-letter code) A-N-S-F-L-X-X-L-R-P-G-N-V-X-R-X-C-S-X-X-V-C-X-F-X-X-A-R-X-I-F-Q-N-T-X-D-T-M-A-F-W-S-K-Y-S-D-G-D-Q-C-E-D-R-P-S-G-S-P-C-D-L-P-C-C-G-R-G-K-C-I-H-G-L-G-G-F-R-C-D-C-A-E-G-W-E-G-R-F-C-L-H-E-V-R-F-S-N-C-S-A-E-B-G-G-C-A-H-Y-C-M-E-E-E-G-R-R-H-C-S-C-A-P-G-Y-R-L-E-D-D-H-Q-L-C-V-S-K-V-T-F-P-C-G-R-L-G-K-R-M-E-K-K-R-K-T-L. The first eleven glutamic acid residues were carboxylated to gamma-carboxyglutamic acid (X). The NH2-terminal, vitamin K-dependent part showed an extensive homology to both prothrombin and factor X, whereas the rest of the chain showed a strong homology to factor X but little similarity to prothrombin.     

Neuropeptide Y: complete amino acid sequence of the brain peptide.

The amino acid sequence of neuropeptide Y, a 36-residue peptide recently isolated from porcine brain, has been determined by using high performance liquid chromatography for separation of its tryptic and chymotryptic fragments and subsequent sequence analysis of the isolated fragments by an improved dansyl Edman subtractive technique. The amino acid sequence of neuropeptide Y has been found to be: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr -Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. Neuropeptide Y has a high degree of sequence homology with peptide YY (70%), the newly isolated porcine intestinal peptide, and pancreatic polypeptide (50%). It is therefore proposed that neuropeptide Y, peptide YY, and pancreatic polypeptide are members of a newly recognized peptide family.     

The complete amino acid sequence of human serum transferrin.

The complete amino acid sequence of human serum transferrin has been determined by aligning the structures of the 10 CNBr fragments. The order of these fragments in the polypeptide chain is deduced from the structures of peptides overlapping methionine residues and other evidence. Human transferrin contains 678 amino acid residues and--including the two asparagine-linked glycans--has an overall molecular weight of 79,550. The polypeptide chain contains two homologous domains consisting of residues 1-336 and 337-678, in which 40% of the residues are identical when aligned by inserting gaps at appropriate positions. Disulfide bond arrangements indicate that there are seven residues between the last half-cystine in the first domain and the first half-cystine in the second domain and therefore, a maximum of seven residues in the region of polypeptide between the two domains. Transferrin--which contains two Fe-binding sites--has clearly evolved by the contiguous duplication of the structural gene for an ancestral protein that had a single Fe-binding site and contained approximately 340 amino acid residues. The two domains show some interesting differences including the presence of both N-linked glycan moieties in the COOH-terminal domain at positions 413 and 610 and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). The locations of residues that may function in Fe-binding are discussed.     

Isolation and complete amino acid sequence of human thymopoietin and splenin.

Human thymopoietin and splenin were isolated from human thymus and spleen, respectively, by monitoring tissue fractionation with a bovine thymopoietin RIA cross-reactive with human thymopoietin and splenin. Bovine thymopoietin and splenin are 49-amino acid polypeptides that differ by only 2 amino acids at positions 34 and 43; the change at position 34 in the active-site region changes the receptor specificities and biological activities. The complete amino acid sequences of purified human thymopoietin and splenin were determined and shown to be 48-amino acid polypeptides differing at four positions. Ten amino acids, constant within each species for thymopoietin and splenin, differ between the human and bovine polypeptides. The pentapeptide active site of thymopoietin (residues 32-36) is constant between the human and bovine thymopoietins, but position 34 in the active site of splenin has changed from glutamic acid in bovine splenin to alanine in human splenin, accounting for the biological activity of the human but not the bovine splenin on the human T-cell line MOLT-4.     

Changes in rat hepatic fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase activity during three days of consumption of a high protein diet or starvation

Changes in plasma glucose, hepatic cyclic AMP, glycogen and fructose 2,6-bisphosphate (F-2,6-P2), and liver 6-phosphofructo-2-kinase (6-PF-2kinase), fructose 2,6-bisphosphatase (F-2,6-P2ase) and phosphoenolpyruvate carboxykinase (PEPCK) activities were examined in rats fed a low protein, high carbohydrate (HC) diet during 3 d of either starvation or feeding a high protein, carbohydrate-free (HP) diet. Under both HP feeding or starvation, liver cyclic AMP increased after 1 d and remained constant thereafter. Whereas plasma glucose was low during starvation, it was unaffected by HP feeding. In both experimental groups, liver glycogen fell after 1 d; thereafter it remained low on starvation, but increased progressively on HP diet reaching 70% of the HC-fed rats value on day 3. Under both experimental conditions, F-2,6-P2 fell 85% after day 1 and was unchanged thereafter. One day after the start of starvation or consumption of the HP diet, 6-PF-2kinase decreased, F-2,6-P2ase increased and 6-PF-2kinase/F-2,6-P2ase ratio decreased, but changes were significantly more important with the HP diet than with starvation. PEPCK activity increased in both experimental conditions, but the increase was greater on the HP diet than on starvation. These findings suggest that during the first 3 d the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation.     

Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation.

We have identified the single phosphorylated tyrosine in p60src, the transforming protein of Rous sarcoma virus, as part of the sequence. NH2-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-COOH. Therefore, this is a sequence that is recognized efficiently by a tyrosine protein kinase in vivo. Phosphorylation of tyrosine in cellular proteins appears to play a role in malignant transformation by four classes of genetically distinct RNA tumor viruses. Phosphorylated tyrosines in several other proteins resemble of the tyrosine in p60src in that they are located 7 residues to the COOH-terminal side of a basic amino acid and either 4 residues to the COOH-terminal side of, or in close proximity to, a glutamic acid residue. Therefore it is possible that these features play a role in the selection of sites of phosphorylation by some tyrosine protein kinases. However, several clear exceptions to this rule exist.     

Structure of canine pulmonary surfactant apoprotein: cDNA and complete amino acid sequence.

The apoproteins of pulmonary surfactant (PSAP) are thought to be critical for normal surfactant function. They bind to surfactant phospholipids and enhance their ability to form surface films in vitro. These acidic glycoproteins have monomeric molecular weights of 36,000, 32,000, and 28,000 (PSAP-36, -32, and -28). Each member of this family of proteins has a similar amino acid composition and their differences in electrophoretic mobility are due in part to glycosylation. We have derived the full amino acid sequence of PSAP-32 from the nucleotide sequence of PSAP cDNA. A cDNA library was prepared from canine lung poly(A)+ RNA and screened with oligonucleotide probes that were based on the NH2-terminal amino acids of PSAP-32 determined by Edman degradation. This protein has the striking feature of collagen-like and non-collagen-like sequences in the same polypeptide chain. There are 24 Gly-Xaa-Yaa triplets, where Yaa is often hydroxyproline. These repeats comprise one-third of PSAP near the NH2 terminus. The remaining two-thirds of PSAP is resistant to bacterial collagenase digestion and contains a possible N-glycosylation site near the carboxyl terminus. The NH2-terminal one-third of PSAP-32 probably contains the cysteine involved in interchain disulfide bonds.